Crown Ethers as Transthyretin Amyloidogenesis ... - ACS Publications

Subscriber access provided by University of Winnipeg Library

Article

Crown Ethers as Transthyretin Amyloidogenesis Inhibitors Takeshi Yokoyama, and Mineyuki Mizuguchi J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01700 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on February 4, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Crown Ethers as Transthyretin Amyloidogenesis Inhibitors

Takeshi Yokoyama1* and Mineyuki Mizuguchi1,2

1Faculty

of Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama

930-0914, Japan 2Graduate

School of Innovative Life Science, University of Toyama, 2630 Sugitani,

Toyama 930-0194, Japan

*Corresponding Author T. Yokoyama, at the address above. Tel: +81 (0)76-434-7570; Fax: +81 (0)76-434-7872; E-mail: [email protected]

ACS Paragon Plus Environment

1

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 36

ABSTRACT Transthyretin (TTR) is a tetrameric protein found in human serum and associated with amyloid diseases. Since the tetramer dissociation and misfolding of the monomer precede amyloid fibril formation, development of a small molecule that binds to TTR and stabilizes the TTR tetramer is an efficient strategy for the treatment of amyloidosis. Here we report our discovery of the anti-TTR amyloidogenesis activities of crown ethers. X-ray crystallographic analysis, binding assay and chemical cross-linking assay showed that 4’-carboxybenzo-18C6 (4) stabilized the TTR tetramer by binding to the allosteric sites on the molecular surface of the TTR tetramer. In addition, 4 synergistically increased the stabilization activity of diflunisal, one of the most potent TTR amyloidogenesis inhibitors. These experimental evidences establish that 4 is a valuable template compound as an allosteric inhibitor of TTR amyloidogenesis.


2

Page 3 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Transthyretin (TTR) is a homo-tetrameric protein that transports the thyroid hormone thyroxine (T4) in blood.1 In addition to its physiological role, TTR is also known as an amyloidogenic protein.2 Human TTR amyloidosis results from deposition of TTR amyloid fibrils in specific tissues, including peripheral nerve, heart and eye tissue. Familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC) are caused by mutations in the TTR gene.3, 4 So far, over 100 mutations in TTR causing amyloidosis have been identified.5 Since TTR amyloid fibrils are believed to be formed through dissociation to the monomer and misfolding of the monomer,6 the amyloidogenic property is closely related to the quaternary structure stability. Indeed, the several TTR variants that are associated with FAP have been shown to exhibit lowered stability in comparison to the wild-type TTR (WT-TTR).7-9 Based on this background, small molecules that kinetically stabilize the TTR tetramer have been developed and used as a successful therapeutic strategy against TTR-associated amyloidosis.10, 11 Specifically, the non-steroidal anti-inflammatory drug diflunisal and novel compound tafamidis have been shown to stabilize the TTR tetramer and inhibit TTR amyloidogenesis.12, 13 Both diflunisal and tafamidis treatments are generally well tolerated.14 For convenience, the individual subunits of the TTR tetramer are designated subunits A to D. The TTR monomer is composed of eight β-strands designated A-H and a short α-helix. The α-helix is called EF-helix, since it is located between the strand E and F. The TTR quaternary structure is assembled mainly by the monomer-monomer interaction (between subunits A and B) and two types of the dimer-dimer interaction (between subunits A and D and between subunits A and C). The dimer-dimer interface is predominantly involved in several hydrogen bonds and hydrophobic interactions between the AB-loop of subunit A and the GH-loop of subunit D. TTR


3


Page 4 of 36

has two funnel-shaped T4-binding sites which are created between subunit A and C. TTR amyloidogenesis inhibitors, such as tafamidis and diflunisal, bind to the T4-binding site and kinetically stabilize the TTR tetramer, resulting in suppression of the amyloid fibril formation.12, 13

The discovery and development of a TTR amyloidogenesis inhibitor is an ongoing project. We considered that 18-crown-6, a classic crown ether, would be a good starting compound for the inhibitor development (Figure 1). Crown ethers are macrocyclic oligomers of ethylene oxide and can capture various cations in their ring depending on the ring size. The cyclic hexamer 18-crown-6 (18C6, 1,4,7,10,13,16-hexaoxacyclooctadecane) is well known as a receptor of protonated amino groups (−NH3+) of lysine residues due to its reasonably sized cavity.15, 16 The human transthyretin monomer possesses 7 lysine residues, and only K15 is located at the T4-binding site, allowing potential electrostatic interactions with kinetic stabilizers. Crystallographic analysis of the TTR-stabilizer complex suggested that formation of a salt bridge with K15 contributes to the stable binding of the stabilizer.17, 18 The Lys-specific molecular tweezer CLR01 has been reported to inhibit the fibrillization of multiple amyloidogenic proteins, including TTR, by binding to lysine residues.19, 20 CLR01 effectively inhibits the nucleation, oligomerization and fibril elongation of amyloidogenic proteins. However, it is conceivable that the T4-binding site of TTR cannot accept the binding of CLR01 due to its high molecular weight. We therefore investigated whether 18C6 binds to K15 at the T4-binding site and stabilizes the TTR tetramer in the manner of a TTR amyloidogenesis inhibitor. Here we describe the potential of crown ethers as TTR amyloidogenesis inhibitors based on analysis by X-ray crystallography, competitive binding assay using fluorescence probes and chemical cross-linking assay. We tested 6 crown ethers: the cyclic tetramer 12-crown-4 (1),


4

Page 5 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pentamer 15-crown-5 (2), 18C6 (3), 4’-carboxybenzo-18C6 (4), dicyclohexa-18C6 (5) and benzo-18C6 (6) (Figure 1).

Figure 1. Chemical structures of the selected crown ethers, diflunisal and tafamidis.


5


Page 6 of 36

RESULTS and DISCUSSION Inhibitory potency against amyloid fibril formation Inhibitory activity of the selected crown ethers against the V30M-mutated TTR (V30MTTR) amyloid fibril formation was investigated by quantification of amyloid fibrils using thioflavin-T as a fluorescence probe (Table 1). V30M is the most common mutation associated with hereditary amyloidosis. Diflunisal, classified as a nonsteroidal anti-inflammatory drug, is one of the most potent TTR amyloidogenesis inhibitors and was used as a positive control in this assay.12 Low-molecular weight linear polyethylene glycols (PEGs), such as PEG200, PEG300 and PEG400, were also used as controls in the experiments, since they are chemically related to crown ethers and possess similar molecular weights. While the addition of 1, 2 or PEGs did not suppress amyloid fibril formation at all, the amyloid fibril formation was slightly but significantly suppressed in the presence of 20 mM compound 3. The formation of TTR amyloid fibril was significantly suppressed to 58% by addition of 2 mM 4 and suppressed to 47% by addition of 10 mM 6, indicating that 4 and 6 are the most potent of the selected crown ethers. These results suggested that a hexameric cyclization is required for the inhibition of TTR amyloid fibril formation and the addition of a phenyl group increased the inhibitory potency.

TTR Stabilization effect of crown ethers Since TTR amyloid fibrils are formed through destabilization of the TTR tetramer, the TTR tetramer-stabilization activity of crown ethers is considered a significantly accurate index of the potential of anti-amyloidogenesis agents. The V30M-TTR tetramer-stabilization activity of


6

Page 7 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


crown ethers was assessed by quantifying the tetramer fractions in a glutaraldehyde cross-linking assay as described previously (Figure 2a, b).21, 22 The TTR tetramer bands were not observed even at 100 mM 3, indicating that the stabilization activity of 3 was too low to detect (Figure 2c). On the other hand, 4 and 6 stabilized the TTR tetramer in a dose-dependent manner. The addition of a phenyl group to 3 appeared to have strengthened the interaction with TTR. The TTR stabilization activities of 1 and 2 were observed at low levels. This may have been due to the non-specificity of the interactions, since the tetramer fractions were not increased in a dosedependent manner. These results indicated that 4 and 6 inhibit the amyloid fibril formation by stabilizing the TTR tetramer.


7


Page 8 of 36

Figure 2. (a) TTR stabilization activities of crown ethers assessed by a glutaraldehyde crosslinking experiment. (a) TTR stabilization activities of diflunisal, 1, 2 and 3. (b) TTR stabilization activities of diflunisal, 4, 5 and 6. M indicates a molecular weight marker and a dash (−) indicates the negative control without a compound. (c) The tetramer fractions are quantified from the SDS-PAGE gels. The tetramer fraction at 10 μM diflunisal is defined as 100%. (d) The TTR stabilization effect of the diflunisal-4 combination dosage was assessed by a glutaraldehyde cross-linking experiment. The lanes pH 8 and pH 4 indicate positive and negative control experiments, respectively. (e) Quantification of the tetramer fractions in the diflunisal-4 combination dosage experiments.


8

Page 9 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Effect of crown ethers on the binding of ANS to TTR 8-anilinonaphthalene-1-sulfonic acid (ANS) has been established as a useful fluorescent probe for the discovery of TTR amyloidogenesis inhibitors. ANS specifically binds to the T4binding site of TTR, and the fluorescence emission of ANS is sufficiently increased upon binding to TTR.23 By utilizing these properties, we determined the half-maximal binding concentration (BC50) for the binding of ANS to TTR and investigated the effect of crown ethers and 150 mM Na+/K+ on BC50 values (Figure 3a). The BC50 value was increased from 3.0 to 12 μM by addition of 5 μM diflunisal under a condition that included 150 mM Na+ (Figure 3b). The BC50 value was also increased to 20 μM by addition of 100 mM 3 under a condition including 150 mM Na+. These results indicated that diflunisal and 3 competitively bind to the T4-binding site with ANS. The increase of the BC50 value for 3 was suppressed to 10 μM under the condition including 150 mM K+, indicating that the chelation of K+ by 3 (presence of K+ at the center of 3) lowers the binding affinity of 3 to TTR. In addition, we investigated the effect of the concentration of NaCl/KCl on the BC50 value for 3 (Table S2). While the BC50 value for 3 was decreased by KCL in a dose-dependent manner, the concentration of NaCl did not influence the binding of ANS to TTR. The finding that the BC50 value under the condition including Na+ was higher than that under the condition including K+ was attributed to the lower affinity of Na+ to 3.24


9


Page 10 of 36

Figure 3. (a) Semilog plot of the ANS binding assay. Continuous lines represent the theoretical curves based on the 4-Parameter Logistic model. The curves of none-Na+, 4-Na+, 3-K+ and 3-Na+ are colored black, blue, green and red, respectively. (b) BC50 values of ANS in the presence of crown ethers and 150 mM NaCl/KCl.

We also determined the BC50 values in the presence of 1 (100 mM), 2 (100 mM), 4 (10 mM), 5 (10 mM) and 6 (10 mM). The presence of 1, 2 and 4 slightly increased the BC50 values irrespective of the presence or absence of the alkali metal ions (Figure 3b). On the other hand, the presence of 5 (5 mM) and 6 (5mM) significantly increased the BC50 values under a condition including Na+ in comparison to those under a condition including K+. These results suggest that


10

Page 11 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5 and 6 bind to the T4-binding site in the same manner as 3, but 1, 2 and 4 do not bind to the T4binding site. Among the selected crown ethers, the TTR stabilization activity of 4 was the highest, although the ANS competitive assay showed that 4 does not bind to the T4-binding site of TTR. We further analyzed the TTR stabilization activity of 4 when administered together with diflunisal (Figure 2d, e). The TTR tetramer fraction was synergistically increased by concurrent use of 4 and diflunisal. The tetramer fractions in the presence of 1 μM and 2 μM diflunisal were 15% and 52%, respectively, and increased to the degree of the positive control (pH 8) by addition of 5 mM 4 (Figure 2e), while the tetramer fraction with a single dosage of 10 mM 4 was 68% (Figure 2c). These results imply that 4 stabilizes the TTR tetramer by binding to a site distinct from the T4-binding site.

Crystal structures of V30M-TTR in complex with crown ethers We first performed the X-ray crystallographic analysis of V30M-mutated TTR (V30MTTR) in the presence of 100 mM 3. V30M-TTR is more amyloidogenic and less stable than WTTTR, although the crystal structures are quite similar.25 In the V30M-TTR structure, the electron density maps of 3 were clearly observed in the T4-binding sites (Figure 4). The nitrogen atom of the amino group of K15 was located at a position offset from the center of 3, and this displacement enabled the amino group of K15 to donate 3 hydrogen bonds to 3 oxygen atoms of 3 (2.5-3.1 Å). The hydrophobic ring of 3 was surrounded by the hydrophobic residues, such as M13, L17 and V121. The van der Waals interactions appeared to stabilize the binding of 3 to TTR.


11


Page 12 of 36

Figure 4. Crystal structure of V30M-TTR in complex with 3 (PDB ID: 6IMX). The cartoon models and carbon atoms of subunits A, B, C and D are colored green, cyan, magenta and yellow, respectively. The carbon atoms of 3 are colored silver. The omit Fourier maps (3.3σ) are shown as a magenta mesh. The close-up view shows the structure around 3 bound to the T4-binding site. Hydrogen bonds are shown as black dashed lines.

In order to elucidate the mechanism underlying the TTR tetramer stabilization by 4, the crystal structure of V30M-TTR in complex with 4 was determined at 1.5 Å resolution (Table S1). The electron density maps of 4 were observed at two distinctive 4 binding-sites located at the molecular surface (sites 1 and 2), but not at the T4-binding site (Figure 5). The carboxyphenyl moiety of 4 was conclusively placed by the omit Fourier map contoured at 2σ (Figure S1). The TTR crystal contained two monomers (subunit A and B) in an asymmetric unit. Since the TTR tetramer can be obtained by 2-fold crystallographic symmetry, the TTR tetramer possesses the


12

Page 13 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pair of site 1 (site 1’) and site 2 (site 2’). Site 1 and 2 are crystallographically identical to site 1’ and 2’, respectively. Site 1 was located at the molecular surface of subunit A. The oxygen atom of 3 accepts a hydrogen bond from N27. Hydrogen bonds among R21(A), Y78(A), and the carboxyl group of 4 were mediated by the 2 water molecules. In particular, R21 is located on the AB loop, which is crucial for the dimer-dimer interaction. The binding of 4 to site 1 likely stabilizes the interactions between the AB and GH loops, resulting in the stabilization of the tetramer. Site 2 is located at the interface of subunits A and D. Hydrogen bonds were formed between the carbonyl group of L82(D) and the oxygen atom of 4 via a water molecule. The carboxyl moiety of 4 was involved in a hydrogen bond network formed by the amide of S85(A), phenol of Y114(A), guanidyl of R21(D), carbonyl of L82(D) and 4 water molecules. These hydrogen bonds appear to bridge adjacent subunits to stabilize the TTR tetramer. Intriguingly, the cyclic polyether moiety of 4 formed no hydrogen bonds with lysine residues. The carboxy phenyl group would be important for the binding of 4 to TTR rather than the cyclic polyether moiety. Since 3 was not observed at these allosteric sites in the V30M-3 complex structure, the addition of the carboxy phenyl group to crown ethers would enable their binding to the allosteric sites.


13


Page 14 of 36

Figure 5. X-ray crystal structures of V30M-TTR complexed with 4 (PDB ID: 6IMY). The structures are colored as described in Figure 4. Hydrogen bonds are indicated as black dashed lines. Water molecules are shown as red spheres. The black dashed box and red dashed box show the close-up views of site 1 and site 2, respectively. The omit Fourier maps (3.0σ) are shown as magenta mesh.


14

Page 15 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The observed binding mode of 4 is reminiscent of the binding of (−)-epigallocatechin gallate (EGCG) to TTR. EGCG, a natural catechin of green tea, was previously shown to stabilize the TTR tetramer by binding to the molecular surface.26-28 One of the EGCG binding sites was located at the dimer-dimer interface between subunit A and D. This EGCG-binding site was close to site 2 for 4, but the interaction patterns were quite different (Figure 6). The galloyl group of EGCG was located at the cleft created by the side chain of R21 (subunit A) and the EFhelix, forming the hydrogen bond to D18 (subunit A). R21 is located at the AB-loop that is predominantly involved in the dimer-dimer contacts. The EF-helix contains H88, which plays a pivotal role for the inter-subunit interactions.29 Therefore, the formation of the hydrogen bond to D18 likely stabilizes the TTR tetramer by stabilizing the AB-loop and the EF-helix.


15


Page 16 of 36

Figure 6. Structural superposition of X-ray crystal structures of V30M-TTR in complex with 4 (PDB ID: 6IMY) and WT-TTR in complex with EGCG (PDB ID: 3NG5). The cartoon models of the subunits A, B, C and D of V30M-TTR-4 are colored green, cyan, magenta and yellow, respectively. The cartoon models, carbon atoms and labels of WTTTR-EGCG are colored orange. The carbon atoms and the labels for V30M-TTR-4 are colored silver and black, respectively. Hydrogen bonds are indicated as black dashed lines. Water molecules are shown as red spheres.


16

Page 17 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The superposition of these two structures leads us to consider that a hybrid compound of 4 and EGCG might be a promising TTR amyloidogenesis inhibitor, since the binding sites of 4 and EGCG are not identical but continuous. The optimization to stabilize the AB-loop and the EF-helix might increase the specificity of the crown ether to TTR. In addition, as described above, the binding of 4 to TTR was not dependent on the binding of the cyclic polyether moiety to lysine residues, and thus the cyclic polyether moiety could be replaced with another functional group during the optimization process. Crown ethers have been shown to be toxic to eukaryotic cell systems, probably due to the ionophore activities of the cyclic polyether moiety.30, 31 The incorporation of cation-complexed crown ethers into the cell membrane disrupts the homeostasis of physiological cations. Therefore, the replacement of the cyclic polyether moiety would putatively reduce the toxicity. Since 4 exhibited millimolar-order inhibitory activity against the TTR amyloidogenesis, an efficient strategy is required for evolution from 4 to a substantial amyloidogenesis inhibitor. The hybridization of 4 and EGCG and optimization to stabilize the AB-loop and the EF-helix would reduce the cytotoxicity and increase the inhibitory potency against the TTR amyloid fibril formation. Conclusion Crown ethers have attracted continuous attention due to their useful cation binding properties. However, although half a century has passed since the discovery of crown ethers in 1967,32 investigation of the biological activity of crown ethers is still in the early stages. In the course of our search for 3-interacting drug-target proteins, we found that the amyloidogenic


17


Page 18 of 36

protein TTR accepts the binding of 3 at the T4-binding site. Crystallographic and ANS competitive-binding analysis clearly showed the binding of 3 to the T4-binding site of TTR (Figure 3 and Figure 4), but the chemical cross-linking experiment showed that 3 did not stabilize the TTR tetramer (Figure 2a). In contrast to 3, 4 was shown not to bind to the T4binding site but to stabilize the TTR tetramer (Figure 3 and Figure 2b). Crystallographic analysis of V30M-TTR in complex with 4 revealed that 4 is an allosteric inhibitor: 4 bound to sites 1 and 2 located at the molecular surface but not to the T4-binding site (Figure 5). The present study thus realized a first step in the development of crown ethers as TTR amyloidogenesis inhibitors.

EXPERIMENTAL Materials V30M-TTR was prepared from an Escherichia coli system as previously described.25 In brief, the transformed cells were cultured at 310 K in Luria Broth medium containing 50 μg/mL ampicillin for 3 hours to an OD600 of 0.6, and then protein expression was induced overnight at 293 K with 0.25 mM isopropyl-β-D-thiogalactopyranoside. The cell pellets were lysed using sonication, and the sonicated cell debris was removed by centrifugation at 12000 rpm for 60 minutes. The resulting supernatant was purified using Ni-affinity chromatography. The elution fraction was dialyzed against a buffer [20 mM Tris-HCl pH 8.0 and 150 mM NaCl]. The protein samples were frozen using liquid nitrogen until use. The protein purity was assessed by SDS-


18

Page 19 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PAGE with Coomassie brilliant blue staining and yielded a single band. The success of crystallization also assured the high protein purity. Crown ethers were purchased from Tokyo Chemical Industry. The product codes of 1, 2, 3, 4, 5 and 6 are C0858, C0859, C0860, C1714, D1668 and B1539, respectively. Compound 4 (lot No. 7LGNL) was supplied in greater than 97% purity with certificate of analysis by gas chromatography, and other crown ethers were supplied in at least 96% purity.

Crystallography Crystals were obtained by the hanging drop vapor-diffusion method at 293K in the presence of 100 mM 3 or 10 mM 4. The crystallization condition was [27% PEG400, 0.1 M MES pH 6.5, 0.4 M CaCl2]. X-ray diffraction data were collected at beamline 5A at the Photon Factory or beamline NW12A at the Photon Factory Advanced Ring in Japan. The diffraction data were processed with XDS.33 The 3D structures and dictionary data for 3 and 4 were generated using the PRODRG server.34 Protein structures were refined using PHENIX.REFINE with manual model building using COOT.35, 36 The final models were validated using the Protein Data Bank validation suite.37 The crystal and refinement data are summarized in Table S1.

Florescence binding assay


19


Page 20 of 36

The binding affinity of ANS with V30M-TTR was determined in the presence of crown ethers. The fluorescence intensities were recorded on a TECAN GENios microplate reader (MTX Lab Systems, USA). The samples contained 2 μM V30M-TTR, 50 mM Tris-HCl pH 7.4, 150 mM NaCl (or KCl), diflunisal/crown ether and ANS. The concentration of diflunisal was 5 μM. The concentrations of 1, 2 and 3 were 100 mM. The concentrations of 4, 5 and 6 were 10 mM. The ANS concentrations were 0.0025, 0.0083, 0.025, 0.083, 0.25, 0.83, 2.5, 8.3, 25 and 83 μM. The excitation and emission wavelengths were 360 and 465 nm, respectively. The halfmaximal binding concentration (BC50) was estimated by curve fitting using a 4-parameter logistic model with the least-squares methods.38

Glutaraldehyde cross-linking assay Glutaraldehyde cross-linking experiments were performed as previously described.22 Briefly, 50 μL of 3.6 μM V30M-TTR in 100 mM NaOAc pH 4.4 was incubated at 310 K for 10 days in the presence of diflunisal or crown ethers. After the incubation, the sample was mixed with 2.5 μL of 25% glutaraldehyde and incubated at RT for 4 minutes. The reaction was terminated by the addition of 5 μL of 7% (w/v) sodium borohydrate in 0.1 M NaOH. The aliquots were then mixed with SDS sample buffer, boiled and resolved by SDS-PAGE followed by silver staining. Quantification of protein bands was carried out using ImageJ (NIH). In the single-compound assay (Figure 2a-c), the concentrations of diflunisal were 4 and 10 μM, the concentrations of 1, 2 and 3 were 40 and 100 mM, and the concentrations of 4, 5 and 6 were 4 and 10 mM. In the diflunisal and 4 combined assay (Figure 2d, e), the diflunisal concentrations


20

Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were 1, 2 and 4 μM in the presence or absence of 5 mM 4. Samples incubated in 0.1 M Tris-HCl pH 8.0 without compounds were used as a positive control, and samples incubated in 100 mM NaOAc pH 4.4 without compounds were used as a negative control.

Acid-mediated aggregation assay The acid-mediated aggregation assay was performed as previously described with some minor modifications.25 In brief, 2 μM V30M-TTR in solution [50 mM sodium acetate pH 4.2] was incubated with the crown ethers at room temperature for 4 days. After the incubation period, the reaction was stopped by addition of 0.2 M Tris-HCl pH 8.0. Fluorescence emission spectra were obtained in the presence of 20 μM thioflavin-T with excitation and emission wavelengths of 440 and 484 nm, respectively.

Acknowledgements We gratefully acknowledge access to the synchrotron radiation facility at PF, Japan. This work was supported by the Takeda Science Foundation, Japan and by a JSPS KAKENHI grant (Project No. 16K08193). AUTHOR INFORMATION Corresponding Author * T. Yokoyama;


21


Page 22 of 36

Tel: +81 (0)76-434-7570; Fax: +81 (0)76-434-7872; E-mail: [email protected] Notes The authors declare no competing financial interest. ABBREVIATIONS TTR, transthyretin; T4, thyroxine; FAP, familial amyloid polyneuropathy; FAC, familial amyloid cardiomyopathy; WT-TTR, wild-type TTR; 18C6, 1,4,7,10,13,16hexaoxacyclooctadecane; V30M-mutated TTR, V30M-TTR; MTH1, MutT homologue 1; EphA3, ephrin type-A receptor 3; BRD4, bromodomain-containing protein 4; BRD1, bromodomaincontaining protein 1; DOT1L, disruptor of telomeric silencing 1-like; DAPK1, death-associated protein kinase 1; ANS, 8-anilinonaphthalene-1-sulfonic acid; BC50, half-maximal binding concentration; EGCG, (−)-epigallocatechin gallate Accession Codes

The coordinates and structure factors of V30M-TTR-3 and V30M-TTR-4 have been deposited in the Protein Data Bank under the PDB codes 6IMX and 6IMY, respectively. The authors will release the atomic coordinates and experimental data upon publication of the article. Supporting Information The following Supporting Information is available free of charge on the ACS Publications website.


22

Page 23 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Molecular formula strings of all compounds (CSV) Statistics on the X-ray and refinement data and omit difference Fourier map of 4 (PDF)

BC50 values for compound 3 in the presence of various concentrations of NaCl or KCl (PDF)

REFERENCES 1.

Bartalena, L.; Robbins, J. Thyroid-Hormone Transport Proteins. Clinics in Laboratory

Medicine 1993, 13, 583-598. 2.

Cornwell, G. G.; Sletten, K.; Johansson, B.; Westermark, P. Evidence That the Amyloid

Fibril Protein in Senile Systemic Amyloidosis Is Derived from Normal Prealbumin. Biochemical and Biophysical Research Communications 1988, 154, 648-653. 3.

Plante-Bordeneuve, V. Transthyretin Familial Amyloid Polyneuropathy: An Update.

Journal of Neurology 2018, 265, 976-983. 4.

Siddiqi, O. K.; Ruberg, F. L. Cardiac Amyloidosis: An Update on Pathophysiology,

Diagnosis, and Treatment. Trends in Cardiovascular Medicine 2018, 28, 10-21. 5.

Sekijima, Y.; Yoshida, K.; Tokuda, T.; Ikeda, S. Familial Transthyretin Amyloidosis.

GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2018. 2001, Available from: https://www.ncbi.nlm.nih.gov/books/NBK1194/.


23


6.

Page 24 of 36

Kelly, J. W.; Colon, W.; Lai, Z. H.; Lashuel, H. A.; McCulloch, J.; McCutchen, S. L.;

Miroy, G. J.; Peterson, S. A. Transthyretin Quaternary and Tertiary Structural Changes Facilitate Misassembly into Amyloid. Advances in Protein Chemistry, Vol 50 1997, 50, 161-181. 7.

Niraula, T. N.; Haraoka, K.; Ando, Y.; Li, H.; Yamada, H.; Akasaka, K. Decreased

Thermodynamic Stability as a Crucial Factor for Familial Amyloidotic Polyneuropathy. Journal of Molecular Biology 2002, 320, 333-342. 8.

Quintas, A.; Saraiva, M. J. M.; Brito, R. M. M. The Amyloidogenic Potential of

Transthyretin Variants Correlates with Their Tendency to Aggregate in Solution. Febs Letters 1997, 418, 297-300. 9.

Shinohara, Y.; Mizuguchi, M.; Matsubara, K.; Takeuchi, M.; Matsuura, A.; Aoki, T.;

Igarashi, K.; Nagadome, H.; Terada, Y.; Kawano, K. Biophysical Analyses of the Transthyretin Variants, Tyr114his and Tyr116ser, Associated with Familial Amyloidotic Polyneuropathy. Biochemistry 2003, 42, 15053-15060. 10.

Ueda, M.; Ando, Y. Recent Advances in Transthyretin Amyloidosis Therapy. Transl

Neurodegener 2014, 3, 19. 11.

Yokoyama, T.; Mizuguchi, M. Inhibition of the Amyloidogenesis of Transthyretin by

Natural Products and Synthetic Compounds. Biological & Pharmaceutical Bulletin 2018, 41, 979-984. 12.

Munro, S. L.; Lim, C. F.; Hall, J. G.; Barlow, J. W.; Craik, D. J.; Topliss, D. J.; Stockigt,

J. R. Drug Competition for Thyroxine Binding to Transthyretin (Prealbumin) - Comparison with Effects on Thyroxine-Binding Globulin. Journal of Clinical Endocrinology & Metabolism 1989, 68, 1141-1147.


24

Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


13.

Bulawa, C. E.; Connelly, S.; Devit, M.; Wang, L.; Weigel, C.; Fleming, J. A.; Packman,

J.; Powers, E. T.; Wiseman, R. L.; Foss, T. R.; Wilson, I. A.; Kelly, J. W.; Labaudiniere, R. Tafamidis, a Potent and Selective Transthyretin Kinetic Stabilizer That Inhibits the Amyloid Cascade. Proceedings of the National Academy of Sciences of the United States of America 2012, 109, 9629-9634. 14.

Jono, H.; Ando, Y. Towards Targets and Treatments in Transthyretin Amyloidosis.

Expert Opinion on Orphan Drugs 2017, 5, 691-699. 15.

Oshima, T.; Suetsugu, A.; Baba, Y. Extraction and Separation of a Lysine-Rich Protein

by Formation of Supramolecule between Crown Ether and Protein in Aqueous Two-Phase System. Analytica Chimica Acta 2010, 674, 211-219. 16.

Lee, C. C.; Maestre-Reyna, M.; Hsu, K. C.; Wang, H. C.; Liu, C. I.; Jeng, W. Y.; Lin, L.

L.; Wood, R.; Chou, C. C.; Yang, J. M.; Wang, A. H. J. Crowning Proteins: Modulating the Protein Surface Properties Using Crown Ethers. Angewandte Chemie-International Edition 2014, 53, 13054-13058. 17.

Klabunde, T.; Petrassi, H. M.; Oza, V. B.; Raman, P.; Kelly, J. W.; Sacchettini, J. C.

Rational Design of Potent Human Transthyretin Amyloid Disease Inhibitors. Nature Structural Biology 2000, 7, 312-321. 18.

Tomar, D.; Khan, T.; Singh, R. R.; Mishra, S.; Gupta, S.; Surolia, A.; Salunke, D. M.

Crystallographic Study of Novel Transthyretin Ligands Exhibiting Negative-Cooperativity between Two Thyroxine Binding Sites. PLoS One 2012, 7. 19.

Sinha, S.; Lopes, D. H.; Du, Z.; Pang, E. S.; Shanmugam, A.; Lomakin, A.; Talbiersky,

P.; Tennstaedt, A.; McDaniel, K.; Bakshi, R.; Kuo, P. Y.; Ehrmann, M.; Benedek, G. B.; Loo, J. A.; Klarner, F. G.; Schrader, T.; Wang, C.; Bitan, G. Lysine-Specific Molecular Tweezers Are


25


Page 26 of 36

Broad-Spectrum Inhibitors of Assembly and Toxicity of Amyloid Proteins. J Am Chem Soc 2011, 133, 16958-69. 20.

Ferreira, N.; Pereira-Henriques, A.; Attar, A.; Klarner, F. G.; Schrader, T.; Bitan, G.;

Gales, L.; Saraiva, M. J.; Almeida, M. R. Molecular Tweezers Targeting Transthyretin Amyloidosis. Neurotherapeutics 2014, 11, 450-461. 21.

Colon, W.; Kelly, J. W. Partial Denaturation of Transthyretin Is Sufficient for Amyloid

Fibril Formation Invitro. Biochemistry 1992, 31, 8654-8660. 22.

Yokoyama, T.; Ueda, M.; Ando, Y.; Mizuguchi, M. Discovery of Gamma-Mangostin as

an Amyloidogenesis Inhibitor. Scientific Reports 2015, 5. 23.

Yokoyama, T.; Kosaka, Y.; Mizuguchi, M. Inhibitory Activities of Propolis and Its

Promising Component, Caffeic Acid Phenethyl Ester, against Amyloidogenesis of Human Transthyretin. Journal of Medicinal Chemistry 2014, 57, 8928-8935. 24.

Gokel, G. W.; Leevy, W. M.; Weber, M. E. Crown Ethers: Sensors for Ions and

Molecular Scaffolds for Materials and Biological Models. Chemical Reviews 2004, 104, 27232750. 25.

Yokoyama, T.; Kosaka, Y.; Mizuguchi, M. Crystal Structures of Human Transthyretin

Complexed with Glabridin. Journal of Medicinal Chemistry 2014, 57, 1090-1096. 26.

Ferreira, N.; Cardoso, I.; Domingues, M. R.; Vitorino, R.; Bastos, M.; Bai, G. Y.; Saraiva,

M. J.; Almeida, M. R. Binding of Epigallocatechin-3-Gallate to Transthyretin Modulates Its Amyloidogenicity. Febs Letters 2009, 583, 3569-3576. 27.

Miyata, M.; Sato, T.; Kugimiya, M.; Sho, M.; Nakamura, T.; Ikemizu, S.; Chirifu, M.;

Mizuguchi, M.; Nabeshima, Y.; Suwa, Y.; Morioka, H.; Arimori, T.; Suico, M. A.; Shuto, T.; Sako, Y.; Momohara, M.; Koga, T.; Morino-Koga, S.; Yamagata, Y.; Kai, H. The Crystal


26

Page 27 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Structure of the Green Tea Polyphenol (-)-Epigallocatechin Gallate-Transthyretin Complex Reveals a Novel Binding Site Distinct from the Thyroxine Binding Site. Biochemistry 2010, 49, 6104-6114. 28.

Ferreira, N.; Saraiva, M. J.; Almeida, M. R. Natural Polyphenols Inhibit Different Steps

of the Process of Transthyretin (Ttr) Amyloid Fibril Formation. Febs Letters 2011, 585, 24242430. 29.

Yokoyama, T.; Mizuguchi, M.; Nabeshima, Y.; Kusaka, K.; Yamada, T.; Hosoya, T.;

Ohhara, T.; Kurihara, K.; Tomoyori, K.; Tanaka, I.; Niimura, N. Hydrogen-Bond Network and Ph Sensitivity in Transthyretin: Neutron Crystal Structure of Human Transthyretin. Journal of Structural Biology 2012, 177, 283-290. 30.

Kralj, M.; Tusek-Bozic, L.; Frkanec, L. Biomedical Potentials of Crown Ethers:

Prospective Antitumor Agents. Chemmedchem 2008, 3, 1478-1492. 31.

Gumila, C.; Ancelin, M. L.; Delort, A. M.; Jeminet, G.; Vial, H. J. Characterization of the

Potent in Vitro and in Vivo Antimalarial Activities of Ionophore Compounds. Antimicrobial Agents and Chemotherapy 1997, 41, 523-529. 32.

Pedersen, C. J. Cyclic Polyethers and Their Complexes with Metal Salts. Journal of the

American Chemical Society 1967, 89, 2495-2496. 33.

Kabsch, W. Xds. Acta Crystallographica Section D-Biological Crystallography 2010, 66,

125-132. 34.

Schuttelkopf, A. W.; van Aalten, D. M. F. Prodrg: A Tool for High-Throughput

Crystallography of Protein-Ligand Complexes. Acta Crystallographica Section D-Biological Crystallography 2004, 60, 1355-1363.


27


35.

Page 28 of 36

Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.;

Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. Phenix: A Comprehensive Python-Based System for Macromolecular Structure Solution. Acta Crystallographica Section D-Biological Crystallography 2010, 66, 213-221. 36.

Emsley, P.; Cowtan, K. Coot: Model-Building Tools for Molecular Graphics. Acta

Crystallographica Section D-Biological Crystallography 2004, 60, 2126-2132. 37.

Yang, H. W.; Guranovic, V.; Dutta, S.; Feng, Z. K.; Berman, H. M.; Westbrook, J. D.

Automated and Accurate Deposition of Structures Solved by X-Ray Diffraction to the Protein Data Bank. Acta Crystallographica Section D-Biological Crystallography 2004, 60, 1833-1839. 38.

Sebaugh, J. L. Guidelines for Accurate Ec50/Ic50 Estimation. Pharm Stat 2011, 10, 128-

34.


28

Page 29 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Inhibitory activity of crown ethers against the TTR amyloid fibril formation. Compd.

Concentration

Negative control Diflunisal

PEG200

PEG300

PEG400

1 2

3

4

5

6

Amyloid fibril (%) 100 ± 0.97

0.5 μM

80 ± 9.0

1.5 μM

35 ± 1.0

5 μM

3.1 ± 1.6

2 mM

99 ± 1.7

6 mM

99 ± 1.2

20 mM

94 ± 3.2

2 mM

99 ± 0.18

6 mM

102 ± 3.4

20 mM

97 ± 1.5

2 mM

103 ± 1.0

6 mM

102 ± 1.3

20 mM

98 ± 0.56

6 mM

110 ± 7.9

20 mM

99 ± 9.9

6 mM

100 ± 5.8

20 mM

100 ± 3.8

2 mM

100 ± 0.31

6 mM

98 ± 0.70

20 mM

88 ± 5.6

0.2 mM

99 ± 0.25

0.6 mM

92 ± 1.0

2 mM

58 ± 2.4

0.2 mM

100 ± 3.4

0.6 mM

100 ± 2.9

2 mM

110 ± 4.9

10 mM

92 ± 2.0

0.2 mM

100 ± 0.53

0.6 mM

96 ± 4.3

2 mM

74 ± 0.87

10 mM

47 ± 0.39


29


Page 30 of 36

Table of Contents Graphic


30

Page 31 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Chemical structures of the selected crown ethers, diflunisal and tafamidis. 177x80mm (300 x 300 DPI)



Figure 2. (a) TTR stabilization activities of crown ethers assessed by a glutaraldehyde cross-linking experiment. (a) TTR stabilization activities of diflunisal, 1, 2 and 3. (b) TTR stabilization activities of diflunisal, 4, 5 and 6. M indicates a molecular weight marker and a dash (−) indicates the negative control without a compound. (c) The tetramer fractions are quantified from the SDS-PAGE gels. The tetramer fraction at 10 μM diflunisal is defined as 100%. (d) The TTR stabilization effect of the diflunisal-4 combination dosage was assessed by a glutaraldehyde cross-linking experiment. The lanes pH 8 and pH 4 indicate positive and negative control experiments, respectively. (e) Quantification of the tetramer fractions in the diflunisal-4 combination dosage experiments. 190x233mm (300 x 300 DPI)


Page 32 of 36

Page 33 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. (a) Semilog plot of the ANS binding assay. Continuous lines represent the theoretical curves based on the 4-Parameter Logistic model. The curves of none-Na+, 4-Na+, 3-K+ and 3-Na+ are colored black, blue, green and red, respectively. (b) BC50 values of ANS in the presence of crown ethers and 150 mM NaCl/KCl. 187x175mm (300 x 300 DPI)



Figure 4. Crystal structure of V30M-TTR in complex with 3 (PDB ID: 6IMX). The cartoon models and carbon atoms of subunits A, B, C and D are colored green, cyan, magenta and yellow, respectively. The carbon atoms of 3 are colored silver. The omit Fourier maps (3.3σ) are shown as a magenta mesh. The close-up view shows the structure around 3 bound to the T4-binding site. Hydrogen bonds are shown as black dashed lines. 185x102mm (300 x 300 DPI)


Page 34 of 36

Page 35 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. X-ray crystal structures of V30M-TTR complexed with 4 (PDB ID: 6IMY). The structures are colored as described in Figure 4. Hydrogen bonds are indicated as black dashed lines. Water molecules are shown as red spheres. The black dashed box and red dashed box show the close-up views of site 1 and site 2, respectively. The omit Fourier maps (3.0σ) are shown as magenta mesh. 190x220mm (300 x 300 DPI)



Figure 6. Structural superposition of X-ray crystal structures of V30M-TTR in complex with 4 (PDB ID: 6IMY) and WT-TTR in complex with EGCG (PDB ID: 3NG5). The cartoon models of the subunits A, B, C and D of V30M-TTR-4 are colored green, cyan, magenta and yellow, respectively. The cartoon models, carbon atoms and labels of WT-TTR-EGCG are colored orange. The carbon atoms and the labels for V30M-TTR-4 are colored silver and black, respectively. Hydrogen bonds are indicated as black dashed lines. Water molecules are shown as red spheres. 190x166mm (300 x 300 DPI)


Page 36 of 36

Crown Ethers as Transthyretin Amyloidogenesis ... - ACS Publications

Recommend Documents